The stability or integrity of the genomes of eukaryotes is the result of a complex interplay of functions at the center of which is regulation of DNA damage checkpoints and DNA repair [Petrini, J. H., 1999 Amer. J. Hum. Genet., 64:1264–1269]. In eukaryotes, when the DNA is damaged, the cell must first sense that damage is present, then induce cell cycle arrest by activating an evolutionarily conserved DNA damage checkpoint. The checkpoint causes arrest of the cell cycle at the G1/S and G2/M boundaries and activation of DNA repair functions [Elledge, S. J., 1996 Science, 274:1664–1672; Longhese, M. P. et al, 1998 EMBO J., 17:5525–5528; Weinert, T. 1998 Curr. Opin. Genet. Dev., 8:185–193].
Different agents cause different types of DNA damage. Genomic instability, which is a hallmark of neoplastic transformation, may result from defects in the cell cycle checkpoint proteins or DNA repair proteins [Hartwell, L. 1992 Cell 71:543–546; Lengauer, C. et al., 1998 Nature, 396:643–649; and Loeb, L. A., 1991 Cancer Res., 51:3075–3079]. One of the most serious threats to genetic integrity is DNA double-strand breaks (DNA DSBs) which are produced from exogenous agents, such as ionizing radiation, and from errors occurring during normal replication or recombination. DNA DSBs are the most important agents of DNA damage from the cell's perspective, because they are the most difficult to repair.
Considerable information about DNA repair genes and DNA damage checkpoint genes is available. For example, checkpoint proteins are highly conserved and homologues of most are present in S. pombe and higher eukaryotes. Human homologues of S. cerevisiae RAD24, RAD17, MEC3 and DDC1 have been cloned and partially characterized [Bessho, T., and Sancar, A. 2000 J. Biol. Chem., 275:7451–7454; Lieberman, H. B. et al, 1996 Proc. Natl. Acad. Sci. USA, 93:13890–13895; St. Onge, R. P. M. et al, 1999 Mol. Biol. Cell, 10:1985–1995; Volkmer, E., and Karnitz, L. M. 1999 J. Biol. Chem., 274: 567–570]. There are two putative human homologues of Mec1, ATM (ataxia-telangiectasia mutated) [Savitsky, K. et al, 1995 Science, 268:1749–1753] and ATR (AT and rad-related) [Bentley, N. J. et al, 1996 EMBO J., 15:6641–665 1]. In humans, ATM responds to DNA double stranded breaks (DSBs) and when inactivated in patients with ataxia telangiectasia leads to checkpoint defects in G1, S, and G2 [Halazonetis, T. D., and Shiloh, Y. 1999 Biochim Biophys Acta, 1424:R45–55]. Human ATR may mediate the response to DNA damage other than DSBs [Bentley (1996) cited above; Cimprich, K. A. et al, 1996 Proc. Natl. Acad. Sci. USA, 93:2850–2855]. Chk2, the human homologue of S. cerevisiae Rad53, becomes phosphorylated in response to DNA DSBs in an ATM-dependent manner [Blasina, A. et al, 1999 Curr. Biol., 9:1–10; Brown, A. L. et al, 1999 Proc. Natl. Acad. Sci. USA, 96:3745–3750; Matsuoka, S. et al, 1998 Science, 282:1893–1897] leading to stabilization of the tumor suppressor protein p53 and cell cycle arrest in G1 [Chehab, N. H. et al, 2000 Genes Dev., 14:278–288; Matsuoka (1998) cited above]. Germ line mutations in Chk2 are found in Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype typically associated with mutations in p53, suggesting that Chk2 is a tumor suppressor gene and when mutated leads to a predisposition to sarcoma, breast cancer, and brain [Bell, D. W. et al, 1999 Science, 286:2528–2531].
One of the few human homologues that remain to be found is that of budding yeast S. cerevisiae Rad9, which was the first checkpoint protein to be identified [Weinert, T. A., and Hartwell, L. H. 1988 Science, 241:317–322]. Rad9 is a component of the DNA damage checkpoint and is required for cell cycle arrest following genomic insult. Rad9 has two carboxy terminal BRCT (3RCA1 C terminus) domains which are found in many proteins with functions related to the DNA damage response, such as, BRCA1, NBS, XRCC4, DNA ligase 4, PARP, and many others [Bork, P. et al, 1997 Faseb J., 11:68–76; Callebaut, I., and Mornon, J. P. 1997 FEBS—Lett, 400:25–30]. Rad9, along with proteins encoded by genes in the RAD24 epistasis group, including RAD17, RAD24, MEC3, and DDC1 [Longhese (1998), cited above; Paulovich, A. G. et al, 1997 Cell, 88:315–321; Weinert (1998) cited above] are proposed to sense DNA damage and regulate activation and phosphorylation of Mec1, a protein kinase required for subsequent phosphorylation and activation of Rad53/Spk1 and Chk1 kinases. Rad53/Spk1 and Chk1 then phosphorylate proteins that regulate progression through the cell cycle [Sanchez, Y. et al, 1996 Science, 271:357–360; and Sun, Z. et al, 1996 Genes Dev., 10: 395–406].
However, very little is known about the proteins that actually sense DNA damage. The sensing protein varies depending on the type of DNA damage. For example, different proteins are required for activating the DNA damage checkpoint when the cell is exposed to UV light (which induces pyrimidine dimers) than the proteins that are required to activate the DNA damage checkpoint when the cell is exposed to ionizing radiation (which induces DNA strand breaks). Part of the difficulty in identifying sensor proteins is the inability to observe and/or isolate sites of DNA damage, such as DNA DSBs.
Proteins that localize to sites of DNA damage are involved in DNA repair and/or checkpoint control. Thus, one approach useful for visualizing DSBs is by immunofluorescence using antibodies to proteins known to localize to such sites. Such an approach has been employed with the Mre11/Rad50/NBS protein complex, which is involved in DNA repair and checkpoint functions. The Mre11/Rad50/NBS complex forms nuclear foci in response to ionizing radiation that localize to sites of DNA DSBs between four and eight hours after irradiation. Approximately 50% of cells contain on average 12 Mre11 foci per cell 8 hours following 12 Gy gamma irradiation [Maser, R. S. et al, 1997 Mol. Cell. Biol., 17: 6087–6096]. Petrini (1999), cited above exposed partially shielded cells to synchrotron generated ultrasoft x-rays followed by immunofluorescence to probe for Mre11. Mre11 relocalized to the non-shielded areas in a striped pattern corresponding to the regions exposed to X-rays [Nelms, B. E. et al., 1998 Science, 280:590–592]. However, DNA breaks occur immediately after X-rays or gamma irradiation and most of them are repaired well before the four hour time point in which Mre11 foci are evident [Lobrich, M. et al, 1995 Proc. Natl. Acad. Sci. USA, 92:12050–12054]. Also, limited accessibility to a synchrotron irradiator does not allow this approach for visualizing DSB's to be used routinely.
Other proteins that localize to points of DNA damage include BRCA1 [Scully, R. et al, 1997 Cell, 90: 425–435] and Chk2 [Lee, J. S. et al, 2000 Nature, 404:201–204]. BRCA1 and Chk2 form foci predominately in S-phase cells in the absence of DNA damage. These foci disperse within one hour of gamma irradiation and reform approximately 8 hours later [Lee (2000), cited above]. Approximately 10% of cells contain BRCA1 foci that colocalize with the Mre11/Rad50/NBS complex at sites of DNA DSBs [Wang, Y. et al, 2000 Genes Dev., 14:927–939; Zhong, Q. et al, 1999 Science, 285:747–750].
Still other proteins that have been reported to form nuclear foci or redistribute in the nucleus in response to DNA damage are Rad51 [Haaf, T. et al, 1995 Proc. Natl. Acad. Sci. USA, 92:2298–2302] and Rad54 [Tan, T. L. et al, 1999 Curr. Biol., 9:325–328]. Rad51 and Rad54 form nuclear foci in response to ionizing radiation. The foci increase in number with time following treatment with irradiation [Morrison, C. et al, 2000 EMBO J, 19:463–471 and 19(4):786]. Yet another such protein is BLM, which localizes to punctate nuclear structures normally [Gharibyan, V., and Youssoufian, H. 1999 Mol. Carcinog., 26:261–273].
Still other proteins known to localize to sites of DNA DSBs, such as the DNA-PK/Ku complex, ATM and ATR, DNA ligase 4, XRCC4 and PARP, do not form visible nuclear foci in response to DNA damage using immunofluorescence [Lindahl, T., and Wood, R. D. 1999 Science, 286:1897–1905].
Thus, there is at present no method which can use these proteins for the observation and/or isolation of DNA DSBs.
The human p53-binding protein, 53Bp1 was identified in a yeast two-hybrid assay as a protein that binds the p53 tumor suppressor protein. The 53Bp1 was found to bind to the central DNA binding domain of wildtype, but not mutant, p53 and to enhance p53-mediated transcriptional activation of p21. 53Bp1 was proposed to have a role as a transactivator of p53 [Iwabuchi, K. et al, 1994 Proc. Natl. Acad. Sci. USA, 91:6098–6102; Iwabuchi, K. et al, 1998 J. Biol. Chem., 273:26061–26068]. Although 53Bp1 shares no overall homology to other known proteins, the carboxy terminus contains two tandem BRCT domains which are sufficient for binding to p53 [Iwabuchi (1998), cited above]. The nucleotide and protein sequences of 53Bp1 are provided in GENBANK database Accession No. AF078776, submitted Jul. 16, 1998.
There remains a need in the art for methods and compositions for identifying cells and tissues which have sites of DNA damage, e.g., tumor cells, for diagnostic purposes as well as for screening methods for the identification of useful cancer therapeutics.